The experiments described in this proposal are concerned with the elucidation of the mechanism by which vitamin K supports gamma glutamyl carboxylation and with the purification and characterization of the enzyme catalyzing the reaction-the gamma glutamyl carboxylase. Studies on the mechanism include: a. determination of the nature of the activated gamma carbon utilizing Beta-fluorinated glutamyl substrates to measure enzyme and vitamin dependent fluoride elimination (suggestive of a carbanionic mechanism); b. evaluation of the hypotheses that carboxylation proceeds through a vitamin K semiquinone and/or a vitamin K hydroperoxide intermediate; c. determination if products of the carboxylation reaction act to then inhibit the enzyme; d. clarification of the mechanism of action of cyanide on the carboxylase. Three purification strategies will be evaluated. In the first, bovine factor X precursor-carboxylase complexes (from the hepatic endoplasmic reticulum of warfarin-treated calves) will be adsorbed to anti-bovine factor X IgG substituted Sepharose. A variety of elution techniques designed to preserve precursor-carboxylase complexes will be employed (the enzyme is more stable when maintained in the complexed form). In the second, using as antigen carboxylase eluted from precursor carboxylase complexes which were adsorbed to IgG substituted Sepharose, hybridomas will be generated which produce monoclonal antibodies against carboxylase. These antibodies will be linked to Sepharose and carboxylase obtained from normal bovine liver microsomes (i.e., not precursor bound) will be adsorbed to this substituted resin. In the third approach affinity chromatography of carboxylase to peptide-linked vitamin K hydroquinone-Sepharose will be conducted. For both the second and third strategies appropriate elution and storage conditions which preserve carboxylase activity will be employed. While the first strategy will yield purified carboxylase complexed with precursor, the latter two strategies will yield purified carboxylase alone. The purified enzyme will be characterized with respect to physical, chemical, and kinetic properties.